Diagnosis, prevention and treatment of cancer

ABSTRACT

Methods and compositions for the diagnosis, prevention, and treatment of cancer are provided. More particularly, the present invention provides methods and compositions for the diagnosis, prevention, and treatment of cancer through detection and modulation of the expression of TMF/ARA160 and Fer.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to methods and compositions for thediagnosis, prevention, and treatment of cancer in mammals, and inparticular, humans. More particularly, the present invention relates tomethods and compositions for the diagnosis, prevention, and treatment ofcancer through detection and modulation of the expression of TMF/ARA160and Fer.

Cancer is one of the top killing diseases in the western world and vastamounts of effort and financial resources are being invested indeveloping novel therapeutic approaches. However, the need for reliablediagnostic tools, is a rate-limiting step in the successful applicationof a cancer therapy. This is best manifested by the fact that most ofthe currently known markers of cancers, are reliable at the level ofonly 30-50%. Thus the need for new markers that could be reliably usedin the detection of a wide variety of cancers, exists Further, there isa generally accepted need for improved methods of cancer prevention andtreatment, devoid of the well known side effects of current therapies.There is thus a widely recognized need for, and it would be highlyadvantageous to have, methods and compositions for the diagnosis ofcancer that can distinguish the development of the malignant state andfor methods and compositions for prevention and treatment of cancer.

A protein termed TMF or ARA60, which is present in a dormant form innormal mammalian cells has been recently identified (Garcia, J. A. etal. (1992) Proc. Natl. Acad. Sci. USA 89:9372-9376) [OMIM 601126, LocusID 7110, GenBank L01042] Several functions have been attributed to TMF.It was initially identified as a DNA binding protein that preferentiallybinds to the TATA element in the human immunodeficiency 1 (HIV1) longterminal repeat (LTR) Thus, TMF/ARA160 was initially identified as atranscription factor that can suppress transcription of RNA PolymeraseII genes by binding to their TATA box thus giving it the name TATAElement Regulatory Factor (TMF) (Garcia, J. A. et al. (1992) Proc. Natl.Acad. Sci. USA 89:9372-9376). Later, TMF was shown to function as aco-activator of nuclear receptors, particularly the androgen receptor(AR) (Hsiao, P. W. et al. (1999) J. Biol. Chem. 274:22373-22379), a factthat gained it the name androgen receptor coactivator 160 kDa, or ARA160(Hsiao, P.-W. et al. (1999) J. Biol. Chem. 274:22373-22379).

TMF consists of 1093 amino acids with an apparent molecular mass of 160kDa (Hsiao, P. W. at Chang, C. (1999) J. Biol. Chem. 274, 92373-29379;Garcia, J. A., Ou, S. H., Wu, F., Lusis, A. J., Sparkes, R. S. & Gaynor,R. B. (1992) Proc. Natl. Acad. Sci. USA 89, 9372-9376). The central andc-terminal parts of TMF/ARA160 contain coiled coil forming domains (cc)that could mediate the interaction of that protein with other cellularfactors. Using a yeast two hybrid screening system (Schwartz, Y.,Ben-Dor, I., Navon, A., Motro, B. & Nir, U. (1998) FEBS Lett 434,339-345) it has been found that TMF/ARA160 interacts Edith Fer tyrosinekinases and modulates their activities. The Fer and AR binding domainsin TMF/ARA160, overlap and both include cc forming sequences.

Fer (p94^(fer)) is an evolutionarily conserved (Pawson, T., Letwin, K.,Lee, T., Hao, Q.-L., Heisterkamp, N. & Groffen, J. (1989) Mol. Cell.Biol. 9, 5722-5725; Paulson, R., Jackson, J., Immergluck, K. & Bishop,J. M. (1997) Oncogene 14, 641-652) and ubiquitously expressed tyrosinekinase that resides mainly in the cytoplasm and nucleus of expressingcells (Letwin, K., Yee, S.-P. & Pawson, T. (1988) Oncogene 3, 621-627;Hao, Q.-L., Heisterkamp, N. & Groffen, J. (1989) Mol. Cell. Biol. 9,1587-1593; Hao, Q.-L., Ferris, D. K., White, G., Heisterkamp, N. &Groffen, J. (1991) Mol. Cell. Biol. 11, 1180-1183; Kim, L. & Wong, T. W.(1998) J. Biol. Chem. 273, 23542-23548) [OMIM 176942, Locus ID 2241,GenBank J03358]. Fer vas not detected in mourn pert and T cell lines(Halachmy, S., Bern, O., Schreiber, L., Carmel, M., Sharabi, Y., Shoham,J. & Nir, U. (1997) Oncogene 14, 2871-2880).

In the cytoplasm, Fer associates with cell adhesion molecules (Kim, L. &Wong, T. W. (1998) J. Biol. Chem. 273, 23542-23548; Rosato, R.,Veltmaat, J. M., Groffen, J. & Heisterkamp, N. (1998) Mol. Cell. Biol.18, 5762-5770) and Stat3 (Priel-Halachmi, S., Ben-Dor, I., Shpungin, S.,Tennenbaum, T., Molavani, H., Bachrach, M., Salzberg, S. & Nir, U.(2000) J. Biol Chem. 275, 28902-28910) and its kinase activity increasesin growth factor stimulated cells (Kim, L and Wong, T. W (1995)Molecular & Cellular Biology). However, no direct role has beenattributed to Fer in the establishment of adherens junctions or focaladhesions (Craig, A. W., Zimgibl, R., Williams, K. Cole, L. A & Greer,P. A. (2001) Mol. Cell Biol. 21, 603-613), nor was Fer found to beessential for growth factor dependent activation of Stat3. The functionof Fer is redundant in the mouse, and mice devoid of a functional Ferare viable and fertile (Craig, A. W., Zimgibl, R., Williams, K., Cole, LA. & Greer, P. A. (2001) Mol. Cell Biol. 21, 603-613). However, thefunctioning of Fer was found to be pivotal for the proliferation ofmalignant cell lines (Allard, P., Zoubeidi, A, Nguyen, L. T., Tessier,S., Tanguay, S., Chevrette, M., Aprikian, A. & Chevalier, S. (2000) Mol.Cell Endocrinol. 159, 63-77; Orlovsky, K., Ben-Dor, I, Priel-Halachmi,S., Malovany, H. & Nir, U. (2000) Biochemistry 39, 11084-11091) Thus,Fer could be linked to the proliferation of mammalian cells.

A testis specific variant of Fer, termed p51^(ferT), is encoded by analternatively spliced FER transcript (Fischman, K, Edman, J. C.,Shackleford, G. M., Turner, J. A., Rutter, W. J. & Nir, U. (1990) Mol.Cell Biol 10, 146-153; Keshet, E., Itin, A, Fischman, K. & Nir, U.(1990) Mol. Cell. Biol. 10, 5021-5025). Fer and p51^(ferT) shareidentical SH2 and kinase domains but they differ in their N-teminaltails (Hao, Q.-L., Heisterkamp, N. & Groffen, J. (1989) Mol. Cell. Biol.9, 1587-1593; Fischman, K., Edman, J. C., Shackleford, G. M, Turner, J.A., Rutter, W. J. & Nir, U. (1990) Mol. Cell. Biol. 10, 146-153).p51^(ferT) accumulates in late primary spermatocytes (Hazan, B., Bern,O., Carmel, M., Lejbkowicz, F., Goldstein, R. S. & Nir, U. (1993) CellGrowth Differ. 4, 443-449). However the role of that kinase in thespermatogenic process is also not understood (Craig, A. W., Zirngibl,R., Williams, K., Cole, L. A. & Greer, P. A. (2001) Mol. Cell Biol. 21,603-613).

The activities attributed so far to TMF/ARA160 have not been linked tothe Fer tyrosine kinase. Further, it has not heretofore beendemonstrated that levels of expression of TMF/ARA160 can be measured oraltered for diagnosis, prevention, or treatment of cancer. Specificmodulation of expression of TMF/ARA160 and of Fer for prevention andtreatment of cancer have not been developed Consequently there is anunmet need for agents and methods capable of effectively detecting theexpression of TMF/ARA160 for the diagnosis of cancer and for modulationof expression of Fer and of TMF/ARA160 for prevention and treatment ofcancer.

SUMMARY OF THE INVENTION

The present invention is directed to methods and compositions for thediagnosis, prevention, and treatment of cancer. More particularly, thepresent invention provides methods and compositions for the diagnosis,prevention, and treatment of cancer through detection and modulation ofthe expression of TMF/ARA160 and Fer.

According to one aspect of the present invention there is provided anantibody that binds specifically to TMF/ARA160 protein.

According to another aspect of the present invention there is provided akit for detection of TMF/ARA160 protein in a sample, the kit includingthe antibody that binds specifically to TMF/ARA160 protein.

According to yet another aspect of the present invention there isprovided a method of detecting TMF/ARA160 in a biological sample whichincludes providing the biological sample, contacting the biologicalsample with the antibody that binds specifically to TMF/ARA160 protein,and detection binding of the antibody to the TMF/ARA160 protein in thesample.

According to still another aspect of the present invention there isprovided a method of diagnosing a malignant tumor in an individual,which includes providing a biological sample, contacting the biologicalsample with the antibody that binds specifically to TMF/ARA160 protein,and detecting binding of the antibody to a TMF/ARA160 protein in thesample.

According to further features in preferred embodiments of the inventiondescribed below, the method of diagnosing a malignant tumor in anindividual further includes diagnosing the malignant tumor if thebinding of the antibody is absent in the sample.

According to an additional aspect of the present invention there isprovided a method of treating cancer in an individual, which includesincreasing a level of TMF/ARA160 protein in cells or tissues of theindividual.

According to yet an additional aspect of the present invention there isprovided a compound 21 nucleobases in length having the nucleotidesequence corresponding to SEQ ID NO:2 below. According to yet anadditional aspect of the present invention there is provided a compound21 nucleobases in length having the nucleotide sequence corresponding toSEQ ID NO:3 below. According to yet an additional aspect of the presentinvention there is provided a compound 21 nucleobases in length havingthe nucleotide sequence corresponding to SEQ ID NO:5 below. According toyet an additional aspect of the present invention there is provided acompound 21 nucleobases in length having the nucleotide sequencecorresponding to SEQ ID NO:6 below.

According to yet an additional aspect of the present invention there isprovided a short interfering ribonucleic acid molecule, which is aduplex molecule of two compounds, wherein the first compound is thecompound of 21 nucleobases in length having the nucleotide sequencecorresponding to SEQ ID NO:2 below, and the second compound is thecompound of 21 nucleobases in length having the nucleotide sequencecorresponding to SEQ ID NO:3 below. According to yet an additionalaspect of the present invention there is provided a short interferingribonucleic acid molecule, which is a duplex molecule of two compounds,wherein the first compound is the compound of 21 nucleobases in lengthhaving the nucleotide sequence corresponding to SEQ ID NO:5 below, andthe second compound is the compound of 21 nucleobases in length havingthe nucleotide sequence corresponding to SEQ ID NO:6 below.

According to yet an additional aspect of the present invention there isprovided a kit for the treatment of cancer including at least one shortinterfering ribonucleic acid molecule, where the short interferingribonucleic acid molecule is directed against fer mRNA, the shortinterfering ribonucleic acid molecule selected from the siRNAs describedabove.

According to yet an additional aspect of the present invention there isprovided a pharmaceutical preparation comprising at least one shortinterfering ribonucleic acid molecule, where the short interferingribonucleic acid molecule is directed against fer mRNA, the shortinterfering ribonucleic acid molecule is selected from the siRNAsdescribed above along with at least one pharmaceutically acceptablecarrier.

According to yet an additional aspect of the present invention there isprovided a method of treating cancer in an individual, which includesinhibiting the expression of fer in cells or tissues of the individual.

According to further features in preferred embodiments of the inventiondescribed below, the method of treating cancer in an individual, whichincludes inhibiting the expression of fer in cells or tissues of theindividual, includes inhibiting of expression of fer in cells or tissuesof the individual being accomplished by degrading fer mRNA by providinga short interfering ribonucleic acid molecule being directed against fermRNA, the short interfering ribonucleic acid molecule selected from thegroup consisting of the short interfering ribonucleic acid moleculedescribed above.

According to still an additional aspect of the present invention thereis provided a method of treating cancer in an individual animal,particularly a human, which includes inhibiting the expression of a genein cells or tissues of the individual, wherein the inhibiting ofexpression of the gene in cells or tissues of the individual isaccomplished by degrading mRNA corresponding to the gene by providing ashort interfering ribonucleic acid molecule being directed against themRNA.

According to further features in preferred embodiments of the inventiondescribed below, the cancer or malignant tumor is a prostate cancer.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing methods and compositions forthe diagnosis, prevention, and treatment of cancer in manuals, and inparticular, humans. More particularly, the present invention providesmethods and compositions for the diagnosis, prevention, and treatment ofcancer through detection and modulation of the expression of TMF/ARA160and Fer.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is an immunoblot, using the methods and compositions of thepresent invention, demonstrating TMF/ARA160 protein in mammalian celllines;

FIG. 2 is an immunocytochemical analysis, using the methods andcompositions of the present invention, illustrating the subcellularlocalization of TMF/ARA160 in mammalian cells;

FIG. 3 is an immunocytochemical analysis, using the methods andcompositions of the present invention, illustrating that TMF/ARA160co-localizes with the Golgi in mammalian cells;

FIG. 4 is an immunocytochemical analysis, using the methods andcompositions of the present invention, illustrating that TMF/ARA160 isreleased from the Golgi of serum starved C2C12 cells;

FIG. 5 is an immunoprecipitation, using the methods and compositions ofthe present invention, showing that TMF/ARA160 associates with Fer inserum starved C2C12 cells;

FIG. 6 is an immunoprecipitation, using the methods and compositions ofthe present invention, showing that TMF/ARA160 associates with cyclin D1and Stat3 but not with Stat1, in serum starved C2C12 cells;

FIG. 7 is an immunoprecipitation using the methods and compositions ofthe present invention, demonstrating that unbiquitinated proteinsassociate with TMF/ARA160 in serum starved C2C12 cells;

FIG. 8 is an immunoblot, using the methods and compositions of thepresent invention, illustrating TMF/ARA160 levels in benign and inmalignant human menigiomas;

FIG. 9 is an immunoblot, using the methods and compositions of thepresent invention, showing that TMF/ARA160 is not detected humanmalignant prostate xenografts;

FIG. 10 is an immunoblot, using the methods and compositions of thepresent invention, showing the accumulation of TMF/ARA160 in humanprostate cell-lines;

FIG. 11 is an immunocytochemical analysis, using the methods andcompositions of the present invention, of prostate sections;

FIG. 12 illustrates protein levels of various proteins in PC3 cells inculture after siRNA treatment according to the present invention;

FIG. 13 is a graph illustrating the percentage of viable PC3 cells inculture after siRNA treatment according to the present invention;

FIG. 14 is a series of photomicrographs of PC3 cells in culture aftersiRNA treatment according to the present invention (a) compared tocontrol cultures (b and c); and,

FIG. 15 is a graph illustrating tumor growth in a mouse following siRNAtreatment according to the present invention (b) compared to that in acontrol mouse not treated (a).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of methods and compositions which can be usedfor the diagnosis, prevention, and treatment of cancer in mammals, andin particular, humans. Specifically, the present invention includescompositions and methods that can be used for diagnosing cancer byassessing the relative cellular levels of TMF/ARA160 in a biologicalsample. The present invention further includes methods and compositionsthat can be used for the prevention and treatment of cancer byincreasing levels of TMF/ARA160, or reducing levels of fer, in cells andtissues.

The principles and operation of methods and compositions which can beused for the diagnosis, prevention, and treatment of cancer according tothe present invention may be better understood with reference to thedrawings and accompanying descriptions and examples detailedhereinunder.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of construction and the arrangement of the components setforth in the following description or illustrated in the drawings. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

The present invention demonstrates (as shown in the exampleshereinunder) that TMF/ARA160 serves also as a “Gate-Keeper”, or GK, andit antagonizes the establishment of malignant state in mammalian cells.For the purposes of this specification and the accompanying claims, theterms TMF, ARA160, TMF/ARA160, Gate-Keeper, GK, and co-activator areused interchangeably. Further the terms FER tyrosine kinase, FER, andp94fer are also used interchangeably. Consequently, GK interferes withcancer progression and could thus be regarded as a tumor suppressorprotein. It is demonstrated that one mechanism by which TMF/ARA160antagonizes the uncontrolled growth of malignant cells is by recruitingkey proliferation promoting factors like Stat3 and cyclin D1 to theirdegradation For the purposes of this specification and the accompanyingclaims, the terms cancer, malignancy, tumor, and malignant tumor areused interchangeably.

Considering these findings, it was predicted that the cellular effectsof TMF/ARA160 are overridden in established cancer cells or that itsfunction is neutralized in these cells. This could be achieved byantagonizing the activity of TMF/ARA160 or by abolishing itsaccumulation in cancer cells.

To further demonstrate the cellular role of TMF/ARA160 and to exploreits possible link to Fer and Stat3, the subcellular distribution ofTMF/ARA160 was determined and its interactions with other cellularproteins characterized. In the present invention we show that TMF/ARA160is a constituent of a novel regulatory pathway that involves also Ferand Stat3 and is also involved in the modulation of mammaliancell-growth.

The present invention includes compositions and methods for diagnosingcancer by assessing the relative cellular levels of TMF/ARA160 in abiological sample. The present invention is directed toward a method ofdiagnosing cancer in an individual comprising the steps of obtaining abiological sample from an individual and deleting the level ofTMF/ARA160 in the sample. The absence or low level of TMF/ARA160 in thesample is indicative of the presence of cancer in the sample.Preferably, the detection of the level of TMF/ARA160 utilizes antibodyagainst TMF/ARA160. The present invention further features antibodyagainst TMF/ARA160. The invention also encompasses kits for detectingthe presence of TMF/ARA160 in a biological sample. The present inventionfurther includes methods for the prevention and treatment of cancer byincreasing levels of TMF/ARA160 in cells.

Antibodies used in the method of the invention, which can be intactantibodies or fragments thereof, can be polyclonal, or more preferablymonoclonal. The antibody according to the present invention has arelatively-wide applicability to a variety of fields which require thedetection of the polypeptide. When used in labeled immunoassays such asradioimmunoassay, enzyme immunoassay, and fluorescent immunoassay, themonoclonal antibody can qualitatively and quantitatively detect thepolypeptide in samples instantly and accurately. In such assays, themonoclonal antibody is labeled, for example, with radioisotopes, enzymesand/or fluorescent substances prior to use. The antibody specificallyreacts with the polypeptide to exhibit an immunoreaction, and accuratelydetects a slight amount of the polypeptide in samples by measuring thelevel of the immunoreaction for these labeled substances.

The antibodies can be labeled for ease of detection, i.e., directlylabeled through coupling to a detectable substance (e.g., radioactive,enzymatic or fluorescent label) as well as indirectly labeled throughreaction with another reagent that is directly labeled. Examples ofindirect labeling include, for example, detection of a primary antibodyusing a fluorescently labeled secondary antibody. In preferredembodiments, the antibodies, fragments or derivatives are incorporatedinto immunoconjugates consisting of an antibody molecule or bindingregion thereof coupled (i.e., physically linked) to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,radioactive materials metal ions detectable by nuclear magneticresonance, or other tracer molecule can be made by techniques known inthe art. For instance, examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, B-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate; rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of suitable radioactive materials include ¹²⁵I,¹³¹I, ³⁵S, or ³H.

The term “biological sample” is intended to include but not be limitedto, any tissue, cells and biological fluids (including, but not limitedto serum plasma, blood, lymph, cerebrospinal fluid, urine, fluid frombody cavities such as the peritoneal space [ascites], pleural,pericardial, etc., cystic fluid, and exudates) isolated from a subject,including those obtained as surgical and biopsy samples, or fromprocedures such as washings of body cavities, as well as tissues, cellsanal fluids present in vivo within the subject. Surgical and biopsysamples can be procured and samples stored under standard conditions toprevent degradation until the detection method can be performed. Suchsamples include preservation methods such as paraffination.

The detection methods of the invention can be used inimmunohistochemical staining of tissue samples (also known asimmunocytochemical analysis) in order to evaluate the abundance ofTMF/ARA160, or used diagnostically, e.g., in immunoassays, as part of aclinical testing procedure. Such immunoassays include, but are notlimited to, competitive and non-competitive assay systems usingtechniques such as radioimmunoassays, ELISA, “sandwich” immunoassays,precipitin reactions, gel diffusion precipitin reactions,immunoradiometric assays, agglutination assays, complement fixationassays, immunoradiometric assays, fluorescent immunoassays, protein Aimmunoassays, and immunoelectrophoretic assays, to name but a few.

Such measurements can be useful in predictive evaluations of the onsetor progression of cancer. Likewise, the ability to monitor the level ofthis protein in an individual can allow determination of the efficacy ofa given treatment regimen for an individual afflicted with such adisorder.

The invention also encompasses kits for detecting the presence ofTMF/ARA160 in a biological sample (e.g., cells, tissue, surgical orbiopsy specimens, plasma, serum, urine or other biological sample asdefined above). The kits include at least one reagent for detecting thepresence of TMF/ARA160 in a biological sample. For example, the kit cancomprise a labeled or labelable antibody, fragment or derivative whichis capable of detecting TMF/ARA160 in a biological sample; means fordetermining the amount of the protein in the sample; and means forcomparing the amount of TMF/ARA160 in the sample with a standard (e.g.,purified protein) or control sample of normal tissue. The kit mayinclude suitable fluids such as buffers, one or more samplecompartments, standard or control samples, and the like. The kit can bepackaged in a suitable container, which can also include instructionsfor using the kit to detect TMF/ARA160.

Moreover, while the specific examples described herein are from arabbit, this is not meant to be a limitation. The antibodies of theinvention having the desired specificity, whether from a rabbit source,other mammalian source including mouse, rat, sheep, human, or othersources, or combinations thereof, are included within the scope of thisinvention.

The antibodies can be used for the detection and/or enumeration byindirect staining of cells and tissues in normal individuals or indisease states, for example, by fluorescence microscopy, flow cytometry,immunoperoxidase, or other indirect methodologies.

To analyze the relative cellular levels of TMF/ARA160 in normal and inmalignant cells, specific antibodies against the TMF/ARA160 protein(αTMF/ARA160) [α is used throughout to indicate anti-] are raised, whichare used as an efficient analytical tool in the study of that factor.Examples One and Two hereinunder including FIGS. 1-7 and 8-11 givefurther details.

The findings described hereinunder in Examples 1 and 2, and in FIGS.1-11, clearly demonstrate that the absence of TMF/ARA160 or itsaccumulation at relatively low levels, are directly linked to the onsetor progression of the malignant state in mammalian cells. The existenceof that phenomena in solid tumors which were derived from threedifferent tissue, turns the impaired accumulation of TMF/ARA160 into apotential general marker for the onset or progression of cancer. Theability to detect TMF/ARA160 by using assays such as preferably eitherWestern blot or immunohistochemical (see Example one,) analysis allowsassessment of the level of TMF/ARA160, at the multi- and single celllevels, by applying the affinity purified αTMF/ARA160 antibodies. Thisenables the reliable and simple test for early detection of cancercells, for differentiating malignant from benign cells and for easyfollow-up of tumor progression or regression, upon various cancertherapies TMF/ARA160 further serves as a predictive marker forenvisaging the susceptibility of a given tumor to various therapeuticapproaches. Moreover, the relative level of TMF/ARA160 serves as aprognostic tool with which one is able to assess the characteristics(growth rate, metastasis, etc.) of a given tumor. The potentialapplication of the TMF/ARA160 marker is extremely beneficial in highlylethal cancers like pancreatic adenocarcinoma, where tumor specificmarkers are urgently needed.

Activation of the TMF/ARA160 System—The present invention furtherincludes methods for the prevention and treatment of cancer byincreasing levels of TMF/ARA160 in cells. Such increase in TMF/ARA160level can be achieved by activation of the TMF/ARA160 gene or othermethods that increase the levels of TMF/ARA160 protein in the cell. Theinvention features a method of treating cancer in a patient, e.g., amammalian such as a human, by administering to the mammal a compound inan amount effective to increase the level of expression or activity ofthe to TMF/ARA160 gene transcript or TMF/ARA160 gene product to a leveleffective to treat the cancer. In this method the compound can be anucleic acid whose administration results in an increase in the level ofexpression of the TMF/ARA160 gene thereby ameliorating symptoms of thecancer. In another aspect, the invention includes a method forinhibiting tumors in a mammal by administering to the mammal a normalallele of the TMF/ARA160 gene.

Activation of the TMF/ARA160 gene/protein in cancer cells leads to thedownregulation of key proliferation promoting proteins, and to thesubsequent growth arrest and death of the malignant cells. Enforcedactivation of the TMF/ARA160 gene/protein in cancer cells, is preferablyachieved by using two main strategies: a) Activation of the endogenousTMF/ARA60 protein in cancer cells. Some signal-transduction pathwaysinactivate and downregulate the TMF/ARA160 protein in malignant cells.Some of the cellular enzymes which are involved in that inactivationprocess have been identified. At least one of these enzymes was found toplay a pivotal role in the proliferation of cancer cells. However, thefunction of this enzyme is highly redundant in normal cells. Specificinhibiting approaches specifically interfere with the neutralizingactivity of those enzymes, thus leading to the upregulation andactivation of the TMF/ARA160. The end products are drugs that may begiven systematically to patients and will interfere with cellularfactors which downregulate TMF/ARA160. This will consequently lead tothe upregulation and activation of TMF/ARA160 in cancer cells. b) Thesecond strategy relies mainly on the enforced expression of an exogenousTMF/ARA160 protein in cancer cells. This goal is achieved by two maingene therapy approaches. One of them is viral and the other one is nonviral. In the viral approach, the TMF/ARA160 cDNA (or a portion of thegene that directs the production of a normal TMF/ARA160 protein withTMF/ARA160 function) is cloned in viral vectors and the recombinantviruses carrying the TMF/ARA160 cDNA are introduced into tumors. Viralvectors include, but are not limited to adenovirus, adeno-associatedvirus and retrovirus vectors. This leads to the enforced expression ofTMF/ARA160 in cancer cells and to the subsequent death of those cells.In the non viral approach, the TMF/ARA160 cDNA is “trapped” insynthetic, nano-particles (including, but not limited to, liposomes)which serve as vehicles for guided delivery of the TMF/ARA160 cDNA tocancer cells. The TMF/ARA160 cDNA is linked to an appropriate promoterof gene expression and will thus be active upon its penetration to thecells.

The present invention includes approaches for the enforced activation ofTMF/ARA60 (Garcia, J. A. et al (1992) Proc. Natl. Acad Sci. USA89:9372-9376; Hsiao, P.-W. and Chang, C. J. Biol. Chem. (1999)274:22373-22379) in cancer cells. The first approach is based on thefinding that serum starvation leads to the release of TMF/ARA160 fromthe Golgi of cells and to its subsequent activation in the cytoplasm(see Example One, hereinunder). Thus signal transduction factors seem todownregulate the activity of TMF/ARA160 Signal-transduction constituentswhich affect the activity of TMF/ARA160 have been identified. As anon-limiting example, the tyrosine kinase FER (p94fer) is one of thedownregulators of TMF/ARA160. According to the present invention,specific inhibitors to p94fer for subsequent upregulation of TMF/ARA160,in cancer cells are utilized in the prevention or treatment of cancer.Moreover, Fer is essential for the proliferation of cancer cellsindependently of TMF/ARA160 (GK). Fer is highly expressed in all humancancers from both soft and solid tissues. Downregulation of Fer leads togrowth of the growth arrest of prostate cancer cells, as illustratedhereinunder.

A) Developing Inhibitors Directed Toward the Tyrosine Kinase Domain ofp94fer:

1) Combinatorial chemistry is adopted for developing inhibitors thatbind the kinase domain of p94fer and consequently inhibit its kinaseactivity. Similar inhibitor-STI-571- of the oncogenic tyrosine kinasev-abl (which is causal of certain leukemias) was successfully applied inthe treatment of certain human cancers (Marx, J. Science (2001) 292:2231-2233). The drawback of that approach is that it is relativelydifficult to get highly specific inhibitors, which lack side effects.Another, more specific approach is therefore applied in parallel.

2) Applying specific p94^(fer) interfering approaches in malignantcells: Specific low-molecular weight drugs are preferred therapeuticagents in cancer therapy. New potential low-molecular weight moleculetargets are identified that can specifically impair the kinase activityof p94^(fer), in vivo.

The unique N-terminal tail of p94^(fer) drives the oligomerization andautoactivation of the enzyme, in vivo (Orlovsky K, Ben-Dor 1,Priel-Halachmi S, Malovany H. Nir U. Biochemistry 2000 39:11084-11091).This turns this segment into a potential target for specific, p94^(fer)inhibitors. Three CC regions in the p94^(fer) N-terminal tail, werefound to mediate the oligomerization of the enzyme (Ben-Dor I, Bern O,Tennenbaum T, Nir U. Cell Growth Differ 1999;10:113-29; Craig A W,Zimgibl R, Greer P. J Biol Chem 1999;274:19934-42). Out of these, CCI(Craig A W, Zimgibl R, Greer P. J Biol Chem 1999;274:19934-42; OrlovskyK, Ben-Dor I, Priel-Halachmi S, Malovany H, Nir U. Biochemistry 200039:11084-11091) and CCII (Craig A W Zimgibl R. Greer P. J Biol Chem1999;274:19934-42) were found to be essential for the oligomerization ofp94^(fer). Perturbing the structure of either one of these twosubdomains, compromised the ability of p94^(fer) to oligomerize, invivo. Thus, CC domains I and II can serve as interference targets, forinhibiting the oligomerization and autophosphorylation of p94^(fer), invivo. To specifically interfere with the oligomerization of CCI andCCII, two 20 amino acids long polypeptides, one derived from the centralregion of CCI and the other one from CCII (Ben-Dor I, Bern O, TennenbaumT, Nir U. Cell Growth Differ 1999;10:113-29), are synthesized. Thesesynthetic peptides interact and oligomerize with their corresponding, CCsequences in the endogenous p94^(fer), thus interfering with theoligomerization and autophosphorylation of the kinase. Indeed asynthetic peptide directed toward CCI, was shown to interfere with theability of that sub-domain to interact with other CC domains, in vivo(Arregui C, Pathre P, Lilien J, Balsamo J. J Cell Biol2000;149:1263-74.) To increase the membrane permeability of the twopeptides, they may be linked at their N-terminal ends to decanoic acid.This fatty acid was found to effectively assist peptides in crossing thecell membrane, thus avoiding the need for internalization peptidesequences that could affect the structure of the functional peptidesequence (Arregui C, Pathre P, Lilien J, Balsamo J. J Cell Biol2000;149:1263-74). To assess the penetration of the p94^(fer) derivedpeptides, through the cell membrane, the modified peptides arebiotinylated (Arregui C, Pathre P, Lilien J. Balsamo J. J Cell Biol2000;149:1263-74) and incubated (50-100 μm) faith the malignant andnon-malignant breast and prostate cell lines. The subcellularaccumulation and localization of the biotinylated peptides is detectedby using Fluorescein labeled Streptavidin in immunocytochemicalanalysis. As satisfactory permeability is achieved, malignant andnon-malignant cells are incubated with the non-biotinylated peptide. Theeffect of the two synthetic peptides, on the inhibition of p94^(fer)activity and the activation of TMF/ARA160 is determined in cancer cellstreated with 50-100 μm of each of the peptides, for 24, 48 and 72 h.These analyzed parameters are compared to those obtained with anon-relevant 20 amino acid long peptide, derived from the most extremeN-terminal sequence of p94^(fer). That region was not found to beinvolved in the oligomerization of the kinase (Orlovsky K, Ben-Dor I,Priel-Halachmi S, Malovany H, Nir U. Biochemistry 2000 39:11084-11091.)

This is the basis for the design of novel low-molecular weightmolecules, derived from the tested synthetic peptides and whichspecifically interfere with the activity of p94^(fer), in malignantcells.

RNA interference with siRNA: The present application also includescompositions, kits, and methods for the application of short interferingRNA (siRNA) directed toward the Fer mRNA to specifically degrade thatmRNA and consequently downregulate the cellular level of the Ferprotein.

Determining the function of a particular gene is central tounderstanding the genetic and molecular basis of disease. A particularlyeffective method for determining a gene's function is to inactivate, orknock-out, that gene and to then study the effect of that inactivationon the cell or organism. However, classical methods used forinactivation of a specific gene are time consuming, technicallychallenging, costly and in some cases ineffectual and not gene-specific.Recently, a new technique has been developed called RNA interference, orRNAi, which allows for the selective inactivation of a target gene in ahighly specific and effective manner (Sharp, P. A. Genes Dev 15:485-490, 2001; Hannon, G. J. Nature 418: 244-251, 2002; Fire, A. et al.Nature 391: 806-811, 1998). Introduction of double-stranded RNA (dsRNA)corresponding to the sequence of a targeted transcript into a cellcauses the rapid destruction of the targeted gene's messenger RNA(containing the identical sequence to either of the RNA strands in theduplex) thus preventing the production of the protein encoded by thatgene. RNA interference leads to the inhibition of protein expression byutilizing sequence-specific, dsRNA-mediated destruction of the targetmessenger RNA (mRNA). In mammalian cells, the use of long dsRNA (greaterthan 50 bp) has been limited because it also induces a non-specificinhibitory response as part of the interferon pathway, which results ina general inhibition of protein synthesis. Studies indicate this can beavoided by the use of short dsRNAs (of 21-23 bp) called shortinterfering RNAs (siRNAs) (Caplen, N. J., Parrish, S., Imai, F., Fire,A. & Morgan, R. A. Proc Natl Acad Sci USA 98, 9742-9747, 2001; Elbashir,S. M., Lendeckel, W. and Tuschl, T. Genes Dev 15:188-200, 2001,Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., andTuschl, T. Nature 411:494-498, 2001; Brummelkamp, T. R., Bernards, R.,and Aagami, R. Science 296:550-553, 2002; Tuschl, T. NatureBiotechnology 20:446-448, 2002; Paul, C. P., Good, P. D, Winer, I., andEngelke, D. R. Nature Biotechnology 20:505-508, 2002). These siRNAs actcatalytically at sub-molar ratios to cleave greater than 95% of thetarget mRNA in the cell by acting as guides for a cellular enzymecomplex that destroys complementary RNA sequences. The RNA interferenceeffect can be long-lasting and may be detectable after many celldivisions, down regulating gene expression in a sequence-specificmanner. In nature, it is believed that RNAi acts as a natural defenseagainst viral infection, and recent studies have explored its potentialapplication in the control of viral infections such as HIV infection(Lee, N S., Dohjima, T., Bauer, G., et al. Nature Biotechnology20:500-505, 2002; Skipper, M. Nature Rev Gen 3:572-572, 2002). siRNAshave been used heretofore as research reagents by those of ordinaryskill in the art to elucidate the function of particular genes incultured cells. The feasibility of their use in mice in vivo has beendemonstrated for research purposes for studying gene function. The useof RNAi or siRNAs for the prevention or treatment of cancer has notheretofore been described or demonstrated.

The present application includes a method for the treatment of cancerusing the administration of siRNA for modulating the expression ofparticular genes. Specifically, the present application includescompositions, kits, and methods for the application of short interferingUSA (siRNA) directed toward the Fer mRNA to specifically degrade thatmRNA and consequently downregulate the cellular level of the Ferprotein. Use of such compositions and methods led to the growth arrestof prostate cancer cells in in vitro culture and to their subsequentdeath (see Example Three hereinunder). This provides the underlyingbasis for the application of the siRNA approach to the treatment ofcancer in vivo in mammals (see Example Four hereinunder).

The method for the treatment of cancer using the administration of siRNAfor modulating the expression of particular genes includes the deliveryof siRNA duplexes into the tumor cells and tissues, as well as thesurrounding tissues.

For the purposes of this specification and the accompany claims, siRNAis used to refer to RNA duplexes, generally 21-23 nucleotides in length,generally synthetic, which act to degrade mRNA sequences homologous toeither of the RNA strands in the duplex.

A preferred embodiment of the method for the treatment of cancer usingthe administration of siRNA for modulating the expression of particulargenes according to the present invention includes the selection of agene to be targeted for silencing, by degradation of its correspondingmRNA, such that expression of that gene is inhibited. Genes appropriatefor use include those known to be involved in cell growth andproliferation such as proliferation promoting factors, including cellcycle genes, a myriad of which are known (see Vogelstein, B, andKinzler, K. W., The Genetic Basis of Human Cancer McGraw Hill, 2002 forexamples). In a preferred embodiment of the method of the presentinvention the gene for fer is chosen for targeting as illustrated belowin the examples. Alternate genes include those for cell cycle controlgenes such as cdks and cyclins as non-limiting examples.

The method further includes the step of selecting a target sequence inthe target mRNA and design of the siRNA duplexes for the target mRNA.Target sequence selection and siRNA duplex design is based on theguidelines of Tuschl et al., as have become standard in the art (Tuschl,T., P. D. Zamore, R. Lehmann, D. P. Bartel and P. A Sharp Genes Dev 13:3191-3197 (1999); The siRNA user guide[http://www.mpibpc.gwdg.de/abteilungen/100/105/sima.html]; Elbashir S M,Harborth J, Weber K, Tuschl T. Methods Feb; 26(2):199-213, (2002);Technical Bulletin #003-Revision B, Dharmacon Research, Inc. Lafayette,Colo., 2002).

The most efficient silencing is obtained with siRNA duplexes composed of21-nt sense and 21-nt antisense strands, paired in a manner to have a19-nucleotide duplex region and a 2-nt 3′ overhang, (of preferablyeither UU or dTdT) at each 3′ terminus. Symmetric 3′-overhangs ensurethat the sequence-specific endonuclease complexes (siRNPs) are formedwith approximately equal ratios of sense and antisense target RNAcleaving siRNPs. The 3′-overhang in the sense strand provides nocontribution to recognition as it is believed the antisense siRNA strandguides target recognition. Therefore, the UU or dTdT 3′-overhang of theantisense sequences is complementary to the target mRNA but thesymmetrical UU or dTdT 3′-overhang of the sense siRNA oligo does notneed to correspond to the mRNA. The use of deoxythymidines in both3′-overhangs may increase nuclease resistance, although siRNA duplexeswith either UU or dTdT overhangs work equally well. 2′-Deoxynucleotidesin the 3′ overhangs are as efficient as ribonucleotides, but are oftencheaper to synthesize.

The targeted region in the mRNA, and hence the sequence in the siRNAduplex, are chosen using the following guidelines. The open readingframe (ORF) region from the cDNA sequence is recommended for targeting,preferably at least 50 to 100 nucleotides downstream of the start codon,most preferably at least 75-100. Both the 5′ and 3′ untranslated regions(UTRs) and regions near the start codon are not recommended fortargeting as these may be richer in regulatory protein binding sites.UTR-binding proteins and/or translation initiation complexes mayinterfere with binding of the siRNP endonuclease complex.

The sequence of the mRNA or cDNA is searched seeking the sequenceAA(N19)TT. Sequences with approximately 50% G/C-content (30% to 70%) areused. If no suitable sequences are found, the search is extended tosequences AA(N21). The sequence of the sense siRNA corresponds to5′-(N19)dTdT-3′ or N21, respectively. In the latter case, the 3′ end ofthe sense siRNA is converted to dTdT. The rationale for this sequenceconversion is to generate a symmetric duplex with respect to thesequence composition of the sense and antisense 3′ overhangs. It isbelieved that symmetric 3′ overhangs help to ensure that the siRNPs areformed with approximately equal ratios of sense and antisense targetRNA-cleaving siRNPs. The modification of the overhang of the sensesequence of the siRNA duplex is not expected to affect targeted mRNArecognition, as the antisense siRNA strand glides target recognition.

If the target mRNA does not contain a suitable AA(N21) sequence, it isrecommended to search for NA(N21) The sequence of the sense andantisense strand may still be synthesized as 5′ (N19)TT as the sequenceof the 3′ most nucleotide of the antisense siRNA does not appear tocontribute to specificity.

It is further recommended to search the selected siRNA sequence againstEST libraries in appropriate databases (e.g., NCBI BLAST databasesearch) to ensure that only one gene is targeted.

At least one siRNA duplex is used. Although siRNA silencing appears tobe extremely effective by selecting a single target in the mRNA, it ispreferable to design and employ two independent siRNA duplexes tocontrol for specificity of the silencing effect. Studies on thespecificity of target recognition by siRNA duplexes indicate that asingle point mutation located in the paired region of an siRNA duplex issufficient to abolish target mRNA degradation.

The appropriately designed siRNAs are either obtained from commercialsources (such as Dharmacon Research, Lafayette, Colo.; Xergon,Huntsville, Ala.; Ambion, Austin, Tex.) or chemically synthesized usedappropriately protected ribonucleoside phosphoramidites and aconventional DNA/RNA synthesizer according to standard protocols. TheRNA oligonucleotides are fusser 2′ deprotected, desalted and the twostrands annealed, according to manufacturer's specifications orconventional protocols, depending on how the siRNAs are obtained. Allhandling steps are conducted under strict sterile, RNase-freeconditions.

The siRNAs are delivered in various manners, using a transfectioncarrier medium such as a liposome, cationic lipid medium, polyamine,polymer-lipid formulation, receptor targeted molecule, or other suitabletransfection reagent. Such reagents are commercially available (e.g.:TransIT-TKO™, Mirus, Madison, Wis.; Metafectene™, Biontex, Munich,Germany; TransIT-In Vivo™, Mirus, Madison, Wis.; and, Oligofectamine™,Invitrogen, Carlsbad, Calif.) and use for transfection is according tomanufacturers' specifications and instructions for preparation ofsiRNA-complex formation, as well as standardized protocols (e.g.Elbashir S M, Harborth J, Weber K, Tuschl T. Methods Feb. 96(2):199-213,(2002)). Variables such as time and manner of formulation ofsiRNA-carrier complexes is dependent on the specific reagents used andconditions are optimized for the transfection. Variables optimizedinclude, as non-limiting examples, method of mixing (inversion vs.vortexing), formation of complexes in serum-free medium, and optimumreagent and siRNA concentrations.

The transfection reagent may further be a component of a pharmaceuticalformulation or preparation for oral, rectal, ophthalmic, topical(including to mucous membranes), or parenteral administration forassistance in uptake, distribution, absorption and/or delivery of thesiRNA. Such preparations may further be used for administration to therespiratory tract by inhalation or insufflation, to the lungs or viaintratracheal, or intranasal delivery Topical delivery further includesepidermal and transdermal administration. Parenteral administrationincludes intravenous, intrarterial, subcutaneous, intraperitoneal,intracranial, or intramuscular injection or infusion including directinjection into tumor tissue. Pharmaceutical compositions andformulations may contain standard pharmaceutically suitable additionalcomponents including bases, diluents, thickeners, buffers, emulsifiers,and other suitable additives such as are standard in the art, including,but not limited to penetration enhancers, stabilizers, carrier compoundsand other carriers or exipients. A representative United States patentthat teaches pharmaceutical compositions and formulations for deliveryand transfection of oligonucleotides includes, but is not limited toU.S. Pat. No. 6,426,221, which along with the references containedtherein is herein incorporated by reference.

In alternative embodiments siRNA transfer is accomplished ex vivo or invivo using other methods including electroporation, microinjection, orusing specifically designed plasmids, expression constructs, or otherviral and non-viral vectors. Specifically envisaged as being within thescope of the invention is the use of viral and non-viral vectors fordelivery of the siRNA to specific target cells and tissues.

The siRNA is administered to cells and tissues (e g., tumors) eitherdirectly (by injection or topical application, as non-limiting examples)[see Example Four] or by means (intravenous administration, as anon-limiting example) such that the siRNA will be delivered to the tumorsite. Gene silencing has been demonstrated in mouse liver cells aftersiRNA injection into mouse liver in vivo (McCaffrey A P, Meuse L, PhamT-T, Conklin D S, Hannon G J, and Kay M A. Nature 418:38-39, (2002)) andsiRNAs have been demonstrated to be delivered to organs of adult miceafter intramuscular and intravenous injection (Lewis D L, Hagstrom J E,Loomis A G, Wolff J A, Herweijer H. Efficient delivery of siRNA forinhibition of gene expression in postnatal mice. Nat Genet epublicationahead of print; doi:10.1038/ng944, 2002).

The present invention further includes specific oligonucleotides andsiRNA duplexes directed against fer mRNA for use according to themethods of the present invention in the treatment of cancerous tumors(such as prostate cancer as a non-limiting example) as well aspharmaceutical and other compositions comprising the oligonucleotidesand siRNA duplexes and kits including said siRNA oligonucleotides. (seeExamples three and four).

Antisense oligonucleotides: Alternatively, another approach is used inan alternative preferred embodiment of the present invention for thetargeted degradation of the Fer mRNA. Synthetic modified antisensedeoxy-oligonucleotides, which are directed toward defined complementarysequences in the Fer mRNA are designed and introduced to cancer cells.These modified oligo-nucleotides include phosphothioate and/ormethylphosphonate modified deoxy-nucleotides and lead to the degradationof the Fer mRNA sod consequently to the downregulation of the Ferprotein. A representative United States patent that teaches compositionsand methods for use of antisense oligonucleotides for the treatment ofdisease includes, but is not limited to, U.S. Pat. No. 6,426,221, whichalong with the references contained therein is herein incorporated byreference.

B) Introduction of the TMF/ARA160 cDNA into Cancer cells: Viral vectors(including, but not limited to retroviral vectors) are used to allowenforced, stable and long lasting expression of TMF/ARA160 in cancercells. The retroviral vector used, is preferably a broad range vector(Coffin J. Varmus H E (Eds). Retroviruses. New York: Cold Spring HarborLaboratory Press, 1996) into which the TMF/ARA160 cDNA (Garcia, JA. etal. (1992) Proc. Natl. Acad. Sci. USA 89:9372-9376; Hsiao, P. W. andChang, C. J. Biol. Chem. (1999) 274:22373-22379), is transcribed underthe control of the human cytomegalovirus (CMV) early promoter (like thepLNCX2 vector—CLONTECH).

Recombinant retroviruses which carry the TMF/ARA160 cDNA, are used toinfect human cancer cells to enforce ectopic expression of TMF/ARA160 inthe cells. This leads to the growth arrest of the infected cells. In afurther method, the TMF/ARA160 cDNA is expressed tinder the control ofspecific promoters which drive the expression of tumor specificantigens. This restricts the accumulation of the ectopic TMF/ARA160 tocancer cells and avoids the damaging effect of the protein innon-malignant cells.

The present invention thus includes compositions, kits and methods forthe improved diagnosis, prevention, and treatment of cancer.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques described above and beloware those well known and commonly employed in the art Standardtechniques are used for cloning, DNA and RNA isolation, amplificationand purification Generally enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like are performedaccording to the manufacturers specifications. These techniques andvarious other techniques are generally performed according to methodswell known and standard in the art. Such techniques are thoroughlyexplained in the literature. See, for example, “Molecular Cloning: ALaboratory Manual” Sambrook et al., (1989); “Current Protocols inMolecular Biology” Volumes I-III Ausubel, R. M., ed (1994); Ausubel etal., “Current Protocols in Molecular Biology”, John Wiley and Sons,Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”,John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”,Scientific American Books, New York; Birren et al. (eds) “GenomeAnalysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring HarborLaboratory Press, New York (1998); methodologies as set forth in U.S.Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,279,057;“Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J E, ed.(1994); “Culture of Animal Cells—A Manual of Basic Technique” byFreshney, Wiley-Liss, N. Y. (19941), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immnunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); “UsingAntibodies: A Laboratory Manual” (Ed Harlow, David Lane eds., ColdSpring Harbor Laboratory Press (1999)); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4.098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D, and Higgins S. J., eds.(1984); “Animal Cell Culture” Freshney, R. I., ed (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al, “Strategies forProtein Purification and Characterization—A Laboratory Course Manual”CSHL Press (1996); “The Genetic Basis of Human Cancer” Vogelstein, B,and Kinzler, K. W., McGraw Hill (2002); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example One TMF/ARA160 Associates with Fer, Cyclin D1 and ProteasomeDegradable Stat3 in Serum Starved C2C12 Cells

Abbreviations: WGA—Wheat germ hemagglutinin; FCS—Fetal calf serum;BFA—Brefeldin A; DMEM—Dulbecco's modified Eagle's medium; ELISA—Enzymelabeled immunoassay; PBS—Phosphate buffered saline.

Material and Methods

Cell lines C2C12 cells were grown in Dulbecco's modified Eagle's medium(DMEM) supplemented with 15% fetal calf serum (FCS) and 4% glucose. Forgrowth arrest, cells were transferred to medium containing 0.5% FCS and4% glucose. Starvation medium lacked serum but was supplemented with 4%glucose and 2 mM glutamine NIH3T3, COS1 and Hela cells were grown inDMEM containing 10% FCS MCF-7, PC-3 and MDA-MB-231 cells were grown inRPMI supplemented with 10% FCS, 1 mM sodium pyruvate and 2 mM glutamineCells treated with proteasome inhibitor were exposed for 5 hr to 50 mMMG132, before being harvested.

Preparation of αTMF/ARA160 antibodies A cDNA fragment encoding aminoacids 0-268, of the human TMF/ARA160 (N-TMF/ARA160), was flanked by Sph1and Xho1 sites and was inserted between the Sph1 and Sal1 sites in thepQE32, bacterial expression vector (Qiagen). In this vector theexpressed protein is preceded at its N-terminus by six histidineresidues and could thus be purified by immobilized metal affinitychromatography (IMAC). The TMF/ARA160 N-terminal fragment was expressedin E. coli JM109 cells and was then purified by IMAC, using TALON-metalaffinity resin (Clontech) which consists on sepharose-cobalt beads. 200mg of the purified TMF/ARA160 fragment were injected subcutaneously to 3month old female rabbits. This was repeated two more times with 14 dayintervals and the presence of αTMF/ARA160 antibodies in the obtainedsera was determined using ELISA.

For Western blots (immunoblots) and immunocytochemical analysis,TMF/ARA160 anti-sera was affinity purified on a CNBr-activated sepharose(Pharmacia), to which 500 μg of histidine-tagged N-TMF/ARA160 antigenwas covalently linked, according to the manufacturer (Pharmacia)instructions. The anti-serum (4 ml of the second bleeding) was passedthree times through the column to allow the binding of αTMF/ARA160antibodies to the N-TMF/ARA160 antigen. Non-relevant proteins werewashed away with phosphate buffered saline (PBS) and the boundαTMF/ARA160 antibodies were eluted with 100 mM glycine buffer pH 2.5 (20fractions, 0.25 ml each), followed by basic elution with 100 mMtriethylamine pH 11.5 (20 fractions 0.25 ml each). Acidic fractions wereneutralized with 1 M phosphate buffer pH 8 and basic fractions wereneutralized with 1 M phosphate buffer pH 6.8. The titer and specificityof the αTMF/ARA160 antibodies in each factor were determined usingWestern blot analysis and were compared to the titer of the unpurifiedwhole serum.

Immunocytochemical Analysis. Immunocytochemistry was carried outessentially as described before (Priel-Halachmi, S., Ben-Dor, I.,Shpungin, S., Tennenbaum, T., Molavani, H., Bachrach, M., Salzberg, S. &Nir, U. (2000) J. Biol. Chem. 275, 28902-28910; Ben-Dor, I, Bern, O.,Tennenbaum, T. & Nir, U. (1999) Cell Growth Differ. 10, 113-129) ForTMF/ARA160 staining, cells were exposed to 1:150 diluted, affinitypurified αTMF/ARA160 antibodies. The Golgi was stained with 1:1500diluted fluorescein isothiocyanate-labeled wheat germ hemagglutinin(WGA) (Molecular Probes) or with 1:100 diluted αp58 rabbit polyclonalantibodies (Sigma). Rabbit polygonal antibodies were visualized witheither fluorescein isothiocyanate conjugated, or Rhodamin conjugated,(1:200 diluted) donkey anti rabbit secondary antibodies. Nuclei werevisualized by staining the DNA with 0.05 mg/ml propidium iodide or with200 ng/ml solution of Hoechst nuclear dye. Bound fluorophors weredetected with a Bio-Rad MRC 1024 upright confocal microscope with akrypton-argon ion laser Confocal microscope image analysis was performedusing Bio-Rad software, and figures were compiled using the Laser Sharp3.0 software package.

Western blot analysis. Whole cell protein extracts were prepared and 25mg of each sample were resolved by 7% SDS-PAGE, as described before(Priel-Halachmi, S., Ben-Dor, I., Shpungin, S., Tennenbaum, T.,Molavani, H., Bachrach. M., Salzberg S. & Nir, U. (2000) J. Biol. Chem.275, 28902-28910). Electroblotted proteins were detected usingpolyclonal αTMF/ARA160 and αFer (Priel-Halachmi, S., Ben-Dor, I.Shpungin, S., Tennenbaum, T., Molavani, H., Bachrach, M., Salzberg, S. &Nir, U. (2000) J. Biol. Chem. 275, 28902-28910) antibodies; monoclonalαStat3, αStat1 and αcyclin D1 antibodies; and polyclonal αubiquitinantibodies (DAKO).

Immunoprecipitation. Immunoprecipitations were performed basically, asdescribed before (Priel-Halachmi. S., Ben-Dor, I., Shpungin, S.,Tennenbaum, T., Molavani, H., Bachrach, M, Salzberg, S. & Nir, U. (2000)J. Biol. Chem. 275, 28902-28910). In brief extracted proteins (750-1000mg) were incubated overnight at 4 C with 1:50 affinity purifiedαTMF/ARA160 polyclonal antibodies. Antigen-antibody complexes wereprecipitated with protein A-Sepharose for 1 h at 4 C and were washed asdescribed before (Priel-Halachmi, S., Ben-Dor, I., Shpungin, S.,Tennenbaum, T., Molavani, H., Bachrach, M., Salzberg, S. & Nir, U.(2000) J. Biol Chem. 275, 28902-28910). Precipitated proteins were thenresolved by SDS-PAGE, blotted onto nitrocellulose membranes, and reactedwith polyclonal αFer, αubiquitin and αTMF/ARA160 antibodies, ormonoclonal αStat1, αStat3 and αcyclin D1, antibodies.

Results

TMF/ARA160 accumulates in the Golgi of mammalian cells. To furthercharacterize the cellular functions of TMF/ARA160, we have raisedspecific polyclonal antibodies, with which we could follow thesubcellular localization and biochemical characteristics, of thatprotein. Referring now to the drawings, FIG. 1 illustrates theTMF/ARA160 protein in mammalian cell lines by immunoblotting (IB). Wholecell protein extracts from: NIH3T3 (1); PC-3 (2); MCF-7 (3); MDA-MB-231(4) and C2C12 cells (5), were resolved by 7.5% SDS-PAGE. Proteins wereblotted onto a nitrocellulose membrane and were then reacted withαTMF/ARA160 antibodies Migration distances of known protein markers, areshown on the right. Arrows on the left indicate TMF/ARA160. Antibodiesraised against the first 200 amino acids of TMF/ARA160, specificallydetected a 160 kDA protein in all cell lines analyzed (FIG. 1). Howeverin extracts from C2C12 and NIH3T3 cells, additional 110 kDa proteincould be seen (FIG. 1 lanes 1 and 5). The 110 kDa protein represent mostprobably a degradation product of the full size 160 kDa TMF/ARA160protein (Hsiao, P. W. & Chang, C. (1999) J. Biol Chem. 274,22373-22379).

To determine the subcellular distribution of TMF/ARA160, affinitypurified antibodies were applied in an indirect immunocytochemicalassay. FIG. 2 demonstrates the subcellular localization of TMF/ARA160 inmammalian cells. In FIG. 2A, COS1 cells were fixed and incubated faith:pre-immune serum (a); αTMF/ARA160 antibodies (b); and αTMF/ARA160antibodies preincubated with the TMF/ARA160 immunizing antigen (c).Bound antibodies were visualized with Rhodamin-conjugated, donkeyanti-rabbit antibodies. In FIG. 2B, C2C12 (a) and NIH3T3 cells (b) werefixed and stained with propidium iodide (red) and αTMF/ARA160 antibodies(green). Photographs represent confocal laser sections taken 1 μm apart.The original magnification of cells was 600×. Scale bars are in μm.Surprisingly, staining of COS1, NIH3T3 and the myogenic C2C12 cell line,revealed a tightly packed localization of TMF/ARA160, in a region thatwas juxtaposed to the nucleus (FIGS. 2A and B). This stainingrepresented the TMF/ARA160 protein, since it could be competed away by aTMF/ARA160 derived polypeptide, toward which the antibodies were raiseds (FIG. 2A [c]). The tightly packed, perinuclear localization ofTMF/ARA160, resembled the localization of the Golgi apparatus, thatoverlaps the perinuclear localization of the microtubules organizationcenter (moc) (FIG. 3B). To verify the localization of TMF/ARA160 to theGolgi, COS1 and Hela cells were subjected to double-stain,immunocytochemical analysis. The cells were co-stained with αTMF/ARA160antibodies and a fluorescein-labeled WGA, that binds to sugar moietiesin the distal face of the Golgi stack (Tartakoff, A. M. & Vassalli, P.(1983) J. Cell Biol 97, 1243-1248). In parallel, the cells wereco-stained with a specific Golgi marker- the p58 protein which islocated on the microtubule-binding peripheral Golgi membrane (Bloom, G.S. & Brashear, T. A. (1989) J. Biol. Chem. 264, 160S3-16092). WGA andp58 staining overlapped the specific TMF/ARA160 staining in COS1 and inHela cells, and all three stainings were restricted to a the sameperinuclear spot in interphase cells (FIG. 3A and FIGS. 3B a, b and c).However, in mitotic cells TMF/ARA160 was co-dispersed with other Golgicomponents (data not shown) and was less compactly packed also in latediakinesis stages (FIG. 3Ba). Thus TMF/ARA160 is associated with or isstored in the Golgi organelle.

To further support this conclusion, the subcellular localization ofTMF/ARA160, was followed in cells treated with the frugal metabolite,brefeldin A (BFA) that disrupts the Golgi apparatus by interfering withsmall G proteins required for its integrity (Reaves, B. & Banting, G.(1992) J. Cell Biol. 116, 85-94; Lippincott-Schwartz, J., Yuan, L.;Tipper, C, Amherdt, M., Orci, L & Klausner, R. D. (1991) Cell 67,601-616; Robineau, S. Chabre, M. & Antonny, B. (2000) Proc. Natl. AcadSci U.S.A 97, 9913-9918). FIG. 3 further demonstrates that TMF/ARA160co-localizes with the Golgi in mammalian cells. In FIG. 3A, COS1 cellswere fixed and stained with: αTMF/ARA antibodies (a) and WGA (b). Themerged images of -a and b is illustrated in panel c. Cells were stainedwith propidium iodide (red) and αTMF/ARA160 antibodies (green) in paneld. In FIG. 3B, Hela cells were fixed and co-stained with: αtubulin (red)and αTMF/ARA160 (green) antibodies in panels (a) and (b); αp58 (red) andαTMF/ARA160 (green) antibodies in panel (c). BFA treated cells werefixed and co-stained faith αp58 (red) and αTMF/ARA160 antibodies (green)in panels (d) and (e). All nuclei were visualized with Hoechst (blue).The original magnification of the cells was 1000×. In cells exposed toBFA for 4 hr, both the p58 and the TMF/ARA160 staining were less packedand relatively dispersed in the cytoplasm (FIGS. 3B d and e). Thusdisintegration of the Golgi leads to the release of TMF/ARA160 and itsdispersion in the cytoplasm TMF/ARA160 was stored in the Golgithroughout most of the cell cycle stages, and was spread out only duringmitosis, when the Golgi apparatus breaks down (data not shown).

TMF/ARA160 is released from the Golgi of serum deprived C2C12 cells. Theaccumulation of TMF/ARA160 in the Golgi, implies its exclusion from thecell cytoplasm and from the cell nucleus, thus preventing it fromfunctioning as a putative transcription factor. TMF/ARA160 has beenimplicated previously as a general suppressor of genes transcribed byRNA polymerase II (RNA POL.II) (Garcia et al. 1992), and may thus belinked to impaired cell-growth. We therefore checked the subcellulardistribution of TMF/ARA160, in growth arrested cells. Myogenic C2 C12)cells, were grown under both normal and low serum growth conditions,under the latter conditions, the cells undergo gradual differentiationand terminal growth arrest (Craig, A. W., Zirngibl, R., Williams, K.,Cole, L. A & Greer, P. A. (2001) Mol. Cell Biol 21, 603-613). Cells weresubjected to immunocytochemical analysis, before and after beingtransferred to 0.5% FCS, for 48 hr. These experiments were carried outwith low serum rather than a complete lack of serum which led to somecell death, under the immunocytochemistry staining conditions.

FIG. 4 shows that TMF/ARA160 is released from the Golgi of serum starvedC2C12 cells. Actively grossing C2C12 cells (A) and cells grown under lowserum growth conditions (B) were fixed and co-stained with αTMF/ARA160(green) and propidium iodide (red). These photographs represent confocallaser sections taken 1 μm apart. The original magnification of cells was600×. While being stored in the Golgi of actively growing cells,TMF/ARA160 was partially released from that organelle, in cells thatwere grown for 24 hr under low serum growth conditions (data not shown).TMF/ARA160 was further released from the Golgi and was almost fullyspread throughout the cytoplasm, after 48 hr (FIG. 4). Low serum (0.5%FCS) growth conditions did not lead to the disintegration of the Golgi(data not shown), thus suggesting the specific release of TMF/ARA160from the Golgi, under those conditions.

TMF/ARA160 associates with Fer in serum starved C2C12 cells. The releaseof TMF/ARA160 from the Golgi of serum starved cells, and its subsequentspreading in the cytoplasm, should enable the interaction of TMF/ARA160with defined cytoplasmic factors. This process could lead to a newlyactivated function of TMF/ARA160, in growth arrested cells. Onecandidate protein that could potentially interact with TMF/ARA160 in thecytoplasm, is the tyrosine kinase Fer that vas shown to interact withTMF/ARA160, in a yeast two-hybrid screening system (Schwartz, Y.,Ben-Dor, I., Navon, A., Motro, B. & Nir, U. (1998) FEBS Lett. 434,339-345) and which is mainly cytoplasmic in both actively growing and inserum starved C2C12 cells. To test the possible association of these twoproteins in-vivo, TMF/ARA160 was immunoprecipitated from non-starved andfrom serum deprived C2C12 cells by using affinity purified αTMF/ARA160,antibodies. This ensured specific precipitation of TNF/ARA160 and henceother proteins that were co-immunoprecipitated with it. Whole cellextracts were prepared from actively growing C2C12 cultures and fromcells grown without serum for 24, 48 and 72 hr. αTMF/ARA160immunoprecipitates were resolved by SDS-PAGE and were then subjected toαTMF/ARA160 and αFer antibodies in a Western blot analysis.

FIG. 5 is an immunoprecipitation (IP) showing that TMF/ARA160 associateswith Fer in serum starved C2C12 cells. In FIG. 5A, whole cell proteinextracts were prepared from actively growing (lanes 1 and 5) or serumdeprived C2C12 cells (lanes 2-4 and 6-8) which were either untreated(lanes 1-4) or treated with MG132 (lanes 5-8). TMF/ARA160 wasimmunoprecipitated with affinity purified αTMF/ARA160 antibodies.Precipitates were resolved by 7% SDS-PAGE, blotted onto nitrocellulosemembrane and reacted with αTMF/ARA160 (upper panel) or αFER antibodies(lower panel). B. Cellular levels of TMF/ARA160 and Fer, in serumstarved C2C12 cells. Whole cell protein extracts were resolved by 7%SDS-OAGE and exposed in a Western-blot analysis to αTMF/ARA160 (upperpanel) or αFER antibodies (lower panel).

TMF/ARA160 was immunoprecipitated from the different extracts withsimilar efficiency (FIG. 5A). However, obvious levels ofco-immunoprecipitated Fer were detected only after 24 and 48 hr of serumstarvation but not after 72 hr of serum starvation or in proliferating,non starved cells (FIG. 5A lanes 1-4). Thus the Fer kinase associatestransiently with TMF/ARA160, in serum starved C2C12 cells Exposing theTMF/ARA160 immunoprecipitates, to antiphosphotyrosine antibodies (αPT),did not give any signal (data not shown), suggesting that the Ferfraction associated with TMF/ARA160, is not phosphorylated on tyrosine.

Since Fer seemed to be subjected to proteasomal degradation in serumstarved C2C12 cells, immunoprecipitations were performed also fromextracts of cells exposed to the proteasome inhibitor MG132. Ferassociated transiently with TMF/ARA160 in these treated cells as well.However, the levels of the TMF/ARA160 associated Fer increasedsignificantly in cells that were treated with MG132 (FIG. 5A lanes 6 and7). The transient association of TMF/ARA160 and Fer, could reflect thechanges in the accumulation levels of the two proteins, during thestarvation process. We therefore determined the relative cellular levelsof TMF/ARA160 and Fer, in actively growing and in serum starved C2C12cells.

Western blot analysis of whole cell extracts, revealed that while thelevels of TMF/ARA160 were mostly constant (FIG. 5B lanes 1-4) the Ferlevels went down along the starvation process (FIG. 5B lanes 1-4). Thedecline in the Fer level was abolished in cells treated with theproteasome inhibitor MG132 (FIG. 5B lanes 5-8), suggesting that Fer isprone to proteasomal degradation, under those conditions. The transientassociation of Fer and TMF/ARA160 did not reflect, however, the cellularaccumulation profiles of either one of these two proteins, suggestingthat their association is a tightly controlled process.

Thus Fer seems to transiently associate with TMF/ARA160 before beingdirected to proteasomal degradation in serum-starved C2C12 cells.

TMF/ARA160 associates with proteasome degradable Stat3 and with cyclinD1 in serum starved C2C12 cells. The Fer kinase was shown previously toassociate with Stat3 in C2C12 and in NIH3T3 cells (Priel-Halachmi, S.,Ben-Dor, I., Shpungin, S., Tennenbaum, T., Molavani, H., Bachrach, M.,Salzberg, S. & Nir, U. (2000) J. Biol. Chem. 275, 28902-28910). Thisraised the possibility that Stat3 also associates with theTMF/ARA160-Fer complex.

FIG. 6 is an immunoprecipitation showing that TMF/ARA160 associates withcyclin D1 and Stat3 but not with Stat1, in serum starved C2C12 cells.FIG. 6A shows cellular levels of Stat3, cyclin D1 and Stat1, in serumstarved C2C12 cells. Whole cell extracts from actively growing (lanes 1and 5) or serum-starved cells (lanes 2-4 and 6-8) which were eitheruntreated (lanes 1-4) or treated with MG132 (lanes 5-8), were resolvedby 7% SDS-PAGE and exposed to αStat3 (upper panel); αcyclin D1 (middlepanel); or αStat1 antibodies (lower panel), in a Western-blot analysis.FIG. 6B shows that TMF/ARA160 co-immunoprecipitates with Stat3 andcyclin D1 but not with Stat1. TMF/ARA160 was immunoprecipitated from theextracts described in A. Precipitates were resolved by 7% SDS-PAGE andwere reacted with αStat3 (upper panel); αStat1 (middle panel); orαcyclin D1 antibodies (lower panel), in a Western-blot analysis.

Western blot analysis revealed that the accumulation levels of Stat3paralleled the expression profile of Fer, and declined progressively inserum starved C2C12 cells (FIG. 6A lanes 1-4) This decline was not seenin cells exposed to the proteasome inhibitor MG132 (FIG. 6A lanes 5-8),indicating that Stat3 is prone to post-translational degradation in thestarved cells Moreover, high molecular weight forms of Stat3, whichcould represent ubiquitinated Stat3, were detected after 48 hr of serumstarvation (FIG. 6A lanes 6 and 7).

To reveal whether Stat3 associates with TMF/ARA160, proteinsprecipitated with αTMF/ARA160 antibodies were resolved in SDS-PAGE andwere then reacted with αStat3 antibodies, in a Western blot analysis. Aswas seen for the tyrosine kinase Fer, Stat3 did not associate withTMF/ARA160 in actively growing, non-starved cells (FIG. 6B lane 1 and5). However, after 24 and 48 hr of serum starvation, Stat3 associatedtransiently with TMF/ARA160 and this association was barely seen after72 hr of serum starvation (FIG. 6B lanes 4 and 8). The transientassociation of these two proteins increased significantly in cellstreated with MG132, thus suggesting that Stat3 transiently associateswith TMF/ARA160, before being destined to proteasomal degradation. Nosignal was obtained when the TMF/ARA160-Stat3 immunoprecipitates werechallenged with αPT, suggesting that the Stat3 associated withTMF/ARA160 is not phosphorylated on tyrosine

To assess the specificity of the interaction between TMF/ARA160 andStat3, the association of TMF/ARA160 with Stat1 vas analyzed as well.Unlike Stat3 which in many systems is implicated in the promotion ofcell proliferation (Bromberg, J. F., Wrzeszczynska, M. H., Devgan, G.,Zhao, Y., Pestell, R. G., Albanese, C. & Darnell, J. E., Jr. (1999) Cell98, 295-303; Sinibaldi, D., Wharton, W., Turkson, J., Bowman, T.,Pledger, W. J. & Jove, R. (2000) Oncogene 19, 5419-5427), Stat1 islinked mainly to impaired cell growth (Zhou, Y., Wang, S., Gobl, A. &Oberg, K. (2001) Oncology 60, 330-338). In accordance with that notion,the cellular levels of Stat1 increased gradually in C2C12 serum starvedcells (FIG. 6A lanes 1-8). Exposing the TMF/ARA160 immunoprecipitates toαStat1 antibodies in a Western blot analysis, did not reveal anyassociation of Stat1 with TMF/ARA160 in proliferating or in serumstarved C2C12 cells (FIG. 6B lanes 1-8) This proved the specificity ofthe interaction between TMF/ARA160 and Stat3 or Fer, which are linked tothe proliferation of mammalian cells.

To test whether other growth promoting factors could bind to TMF/ARA160,the association of TMF/ARA160 with cyclin D1 (Sinibaldi, D., Wharton,W., Turkson, J., Bowman, T., Pledger, W. J. & Jove, R. (2000) Oncogene19, 5419-5427) was analyzed in serum-starved, C2C12 cells. As was seenfor Stat3, TMF/ARA160 associated with cyclin D1 only at 24 and 48 hrpost starvation, in MG132 treated cells (FIG. 6B lane 4, bottom panel).The cellular level of cyclin D1 was only moderately changing duringC2C12 starvation (FIG. 6A lanes 1-4). Thus TMF/ARA160 bindspreferentially to proliferation-related factors, in serum-starved C2C12cells.

Cytoplasmic TMF/ARA160 associates with ubiquitinated proteins in C2C12cells. The association of TMF/ARA160 with proteasome degradable Stat3,suggested the possible involvement of TMF/ARA160 in the exposure ofStat3 to proteasomal degradation. To confirm the direct link ofTMF/ARA160 to ubiquitin dependent protein degradation, the associationof ubiquitinated proteins with TMF/ARA160 was assessed. TMF/ARA160 wasimmunoprecipitated from both actively growing and serum-stared C2C12cells and the precipitated proteins were resolved by SDS-PAGE.

FIG. 7 is an immunoprecipitation demonstrating that ubiquitinatedproteins associate with TMF/ARA160 in serum starved C2C12 cells.TMF/ARA160 was immunoprecipitated from whole cell protein extracts ofactively grossing (1) or serum starved (2-4) C2C12 cells. Precipitateswere resolved by 7% SDS-PAGE and were reacted with αubiquitin antibodiesin a Western-blot analysis. Migration distances of known molecularweight markers are given on the right. IP: immunoprecipitation; IB:immunoblotting; ab: location of the immunoprecipitating antibodies.

Western blot analysis carried out with αubiquitin antibodies, revealedthe association of ubiquitinated proteins with the TMF/ARA160. Thiscould be seen already in precipitates from actively growing C2C12 cells(FIG. 7 lane 1). However a prominent band of an approximate 90 kDamolecular mass could be clearly seen after 24 hr of serum starvation(FIG. 7 lane 2). This band disappeared after 48 and 72 hr of serumstarvation (FIG. 7 lanes 3 and 4). The estimated size of theubiquitinated protein (90 kDa), suggests that it does not representubiquitinated-TMF/ARA160 species, since ubiquitinated-TMF/ARA160, shouldmigrate as a minimal 160 kDA protein in SDS-PAGE. It is most plausibletherefore that the ubiquitinated 90 kDa band, reflects the transientassociation of TMF zenith another protein.

TMF/ARA160 is a putative transcription factor and co-activator ofnuclear receptors (Garcia, J. A., Ou, S. H. Wu, F., Lusis, A. J.,Sparkes, R. S. & Gaynor, R. B. (1992) Proc. Natl. Acad. Sci. USA 89,9372-9376), whose physiological role has not been conclusively revealedHere we show, that the subcellular localization of TMF/ARA160 is uniqueand very uncommon for a transcription o TMF/ARA160 co-localized with WGAbinding sites in the Golgi, suggesting the accumulation of TMF/ARA160 inthat organelle. Confinement of TMF/ARA160 to the Golgi should excludethat protein from other cellular compartments and components.Furthermore, storage in the Golgi should prevent TMF/ARA160 frominteracting with nuclear receptors and from migrating as their putativeco-activator (Hsiao, P. W. & Chang, C. (1999) J. Biol. Chem. 274,22373-22379) to the nucleus. One would argue that activators of nuclearreceptors such as, glucocorticoids and androgens, could induce therelease of TMF/ARA160 from the Golgi, thus leading to its spreadingthroughout the cytoplasm and its subsequent association with nuclearreceptors. However, we failed to see any effect of dexamethasone on thesubcellular distribution of TMF/ARA160 in C2C12 cells. TMF/ARA160 wasreleased from the Golgi and was spread throughout the cytoplasm, inserum-stained cells. This suggests that signal transduction pathwaysinduced by growth factor stimulation, drive the occlusion of TMF/ARA160in the Golgi. Removal of growth factors from the surrounding of thecells leads therefore to the release of TMF/ARA160 to the cytoplasm.Thus, release of TMF/ARA160 from the Golgi, may be linked to cessationof cell growth.

Spreading of TMF/ARA160 throughout the cytoplasm of C2C12 cells resultsin its association with several cytoplasmic proteins that wereidentified in the current work. These include the tyrosine kinase Fer,cyclin D1 and Stat3. The association of Stat3 faith TMF/ARA160, could bemediated by Fer, since Stat3 was previously shown to associate with Ferin-vivo (Priel-Halachmi, S-, Ben-Dor, I, Shpungin. S., Tennenbaum, T.,Molavani, H., Bachrach, M., Salzberg, S. & Nir, U. (2000) J. Biol. Chem.275, 28902-28910) and Fer was shown to interact directly withTMF/ARA160, in a yeast two-hybrid screening system (Schwartz, Y.,Ben-Dor, I, Navon, A., Motro, B. & Nir, U. (1998) FEBS Lett. 434,339-345) Similarly, the association of Stat3 or cyclin D1 withTMF/ARA160, could also depend on the mutual interaction of these twofactors, which were shown to bind each other, before (Bienvenu, F.,Gascan, H. & Coqueret, O. (2001) J. Biol. Chem. 276, 16840-16847).However, the association of Stat3 with TMF/ARA160 does not seem todepend on Fer or on cyclin D1, since TMF/ARA160 associated with Stat3but not with cyclin D1, in the thymoma derived T127 cells, that lack theFer kinase (Halachmy, S., Bern, O., Schreiber, L., Carmel, M., Sharabi,Y., Shoham, J. & Nir, U. (1997) Oncogene 14, 2871-2880) Thus, Stat3, Ferand cyclin D1 bind TMF/ARA160 either directly or through an unknownmediator.

The levels of Stat3 and Fer dropped significantly in C2C12 cells after72 hr of serum starvation (FIG. 5B and FIG. 6A lanes 1-4). Thisdownregulation was abolished in cells treated with the proteasomeinhibitor GM132 (FIG. 5B and FIG. 6A lanes 5-8s), suggesting that Ferand Stat3 are prone to proteasomal degradation. Proteasomaldownregulation of an inactive Fer enzyme (Craig, A. W., Zimgibl, R.,Williams, K., Cole, L. A. & Greer, P. A. (2001) Mol. Cell Biol. 21,603-613) and of Stat3 (Daino, H., Matsumura, I., Takada, K., Odajima,J., Tanaka, H., Ueda, S., Shibayama, H., Ikeda, H; Hibi, M., Machii, Tet al. (2000) Blood 95, 2577-2585), has been suggested before.Interestingly, both Fer and Stat3 were dephosphorylated on tyrosine andwere both inactive, when they formed a complex with TMF/ARA160.

The association of TMF/ARA160 with Fer and Stat3 was most prominentafter 24 and 48 hr of serum starvation and increased significantly incells treated With GM132. This profile preceded but also overlapped thedownregulation of Fer and Stat3, which was most apparent after 48 and 72hr of serum starvation. Thus, association with TMF/ARA160 could serve asan intermediate and preceding step in the degradation of Stat3 and Fer,under serum starvation. This notion is supported by the fact that theTMF/ARA160 complex contained ubiquitinated proteins after 24 hr of serumstarvation (FIG. 7 lane 2). Another piece of supporting evidence stemsfrom the fact that serum starvation did not lead to the degradation ofStat3, in cells that did not release of TMF/ARA160 from the Golgi.TMF/ARA160 may be a part of a complex, analogous to the ubiquitinationcomplex SCF (DeSalle, L. M. & Pagano, M. (2001) FEBS Left 490, 179-189),that ubiquitinates and recruits a defined set of cellular proteins, tothe proteasome degradation machinery. Sequence analysis did not revealF-box or E3-ligase motifs in TMF/ARA160. However, TMF/ARA160 could serveas a scaffold protein in the assembly of specific ubiquitin-ligase. Asimilar role was attributed to Elongin B C (Conaway, J. W., Kamura, T. &Conawvay, R. C. (1998) Biochim. Biophys. Acta 1377, M49-M54) and to Muf1that like TMF/ARA160 contains leucine rich stretches (Garcia, J. A., On,S. H., Wu, F., Lusis, A. J., Sparkes, R. S. & Gaynor, R. B. (1992) Proc.Natl. Acad Sci. USA 89, 9372-9376) which are essential for itsscaffolding activity (Kamura, T., Burian, D., Yan, Q., Schmidt, S. L,Lane, W. S., Querido, E, Branton, P. E., Shilatifard, A, Conaway, R. C.& Conaway, J. W. (2001) J. Biol. Chem). Interestingly, TMF/ARA160 itselfcontains two potential cyclin degradation boxes that could direct it, toa ubiquitin-mediated proteasomal degradation (Garcia, J. A., Ou, S. H.,Wu, F., Lusis, A, J., Sparkes, R. S. & Gaynor, R. B. (1992) Proc. Nail.Acad. Sci. USA 89, 9372-9376). Hence, the levels of TMF/ARA160 itselfcould be regulated by proteasomal degradation

As mentioned above, cyclin D1 is also a component of the cytoplasmicTMF/ARA160 complexes, in C2C12 cells. However the decline in the cyclinD1 levels in serum-starved C2C12 cells, was-relatively moderate.Therefore one can not exclude the possibility that the decrease in thecyclin D1 levels result from the degradation of Stat3 which serves as anpositive activator of the cyclin D1 gene (Bromberg, J. F., Wrzeszcznska,M. H., Devgan, G., Zhao, Y., Pestell, R. G., Albanese, C. & Darnell. J.E., Jr. (1999) Cell 98, 295-303). The association of TMF/ARA160 withdefined cytoplasmic proteins is specific since TMF/ARA160 did notassociate with Stat1 which is linked to the arrest of cell-growth arrest(Zhou, Y., Wang, S., Gobl, A. & Oberg, K. (2001) Oncology 60, 330-338).Thus TMF/ARA160 associates specifically with proliferation promotingfactors in serum-starved C2C12 cells, and may be involved in theirdownregulation.

C2C12 myoblasts undergo growth arrest and myogenic differentiation uponserum starvation (Craig, A. W., Zimgibl, R., Williams, K., Cole, L A. &Greer, P. A. (2001) Mol. Cell Biol. 21, 603-613) However, the release ofTMF/ARA160 from the Golgi and the down regulation of Fer and Stat3, doesnot seem to be necessarily linked to the myogenic differentiationprocess, since other inducers of myogenic differentiation (Salzberg, S.,Vilchik, S., Cohen, S., Heller, A. & Kronfeld-Kinar, Y. (2000) Exp. CellRes. 254, 45-54; Cohen, R., Valverde, A. M., Benito, M. & Lorenzo, M.(2001) J. Cell Physiol 186, 82-94), did not lead to the time dependentdegradation of these proteins.

It should also be stressed that an efficient release of TMF/ARA160 fromthe Golgi, in all mammalian cell-lines that were grown under low serumconditions was not seen.

TMF/ARA160 seems therefore to be involved in downregulation of key,proliferation promoting factors like Stat3, under defined cellularconditions. One of these could be deprivation of growth factors, thatlike in C2C12 cells, leads to irreversible growth arrest. This may implya novel role for the Golgi apparatus in the modulation of cell-growth.The Golgi, like the mitochondria (Scheffler, I. E. (2001) Adv. DrugDeliv. Rev. 49, 3-26), seems to harbor cellular downregulator(s) thatare released and activated at defined cellular conditions.Interestingly, disintegration of the Golgi by BFA in cancer cells, ledto the degradation of key cell-cycle regulators and consequent celldeath (Chapman, J. R., Tazaki. H., Mallouh, C. & Konno, S. (1999) Mol.Urol. 3, 11-16).

Example Two

Levels of TMF/ARA160 in Normal, Benign and in Malignant Human Tissues

After confirming the quality and reliability of the αTMF/ARA160antibodies, we turned to assess the relative levels of TMF/ARA160 innormal, benign and in malignant human tissues. Whole cell proteinextracts were prepared from normal human tissues, from benign tumors andfrom various human tumors using a Nonidet P-40 (NP40) lysis buffer (1%NP40, 0.5% Sodium deoxycholate, 20 mM Tris.HCl pH 7.5, 150 mM NaCl, 1 mMEDTA and 2 mM Na2VO4), as was described before (Priel-Halachmi, S. etal. (2000) J. Biol. Chem. 275: 28902-28910.) The relative levels ofTMF/ARA160 were then analyzed in benign and in solid malignant humantumors. Protein samples (30 μg) from each preparation were resolved in a7% SDS poly acrylamide gel (SDS-PAGE), blotted onto a nitrocellulosemembrane and were then reacted with the affinity purified αTMF/ARA160antibodies.

FIG. 8 is an immunoblot illustrating TMF/ARA160 levels in benign and inmalignant human menigiomas. 30 mg protein from: benign (lanes 1-3) andfrom malignant menigiomas (lanes 4-7), were resolved by 7% SDS-PAGE andreacted with αTMF/ARA160 (upper panel) or with αFER(p94fer) (lowerpanel), antibodies. Two forms of TMF/ARA160 could be seen in theanalyzed samples (indicated by two arrows on the right side of the upperpanel).

While three benign human menigiomas accumulated relatively high levelsof TMF/ARA160 (FIG. 8 upper panel lanes 1-3), the levels of the proteinwere significantly lower in 5 malignant menigiomas analyzed (FIG. 8upper panel, lanes 4-7). Similarly, very low levels of TMF/ARA160 wereseen also in two malignant human glioblastomas that were analyzedInterestingly the levels of the tyrosine kinase p94fer which was foundto be essential for the proliferation of cancer cells (Allard, P. et al.(2000) Mol. Cell. Endocrinol. 159:63-77; Orlovsky, K. et al. (2000)Biochemistry 39:11084-11091), were similar in both benign and inmalignant tumors (FIG. 8 lower panel).

To further extend our analysis, the levels of TMF/ARA160 were determinedin four human xenografts which were derived from four independentprostate tumors. FIG. 9 shows that TMF/ARA160 is not detected humanmalignant prostate xenografts. Proteins from five independent prostatecancer xenografts (lanes 1-5) and from the myogenic cell line C2C12(lane 6), were resolved in SDS-PAGE and were then reacted withαTMF/ARA160 (upper panel) or with a αFER antibodies (lower panel). Whilethe tyrosine kinase p94fer was clearly detected in all the xenografts(FIG. 9 lower panel), no TMF/ARA160 could be found in the analyzedpreparations (FIG. 9 lanes 1-5, upper panel). To compare the levels ofTMF/ARA160 in malignant prostate cells to its level in transformed butnon-malignant prostate cells, whole cell protein extracts were preparedfrom four different prostate cell lines. These were the benign prostatehyperplasia cell line-BPH1, the androgen sensitive and PSA producingprostate cancer cell line LNCAP which was derived from a metastasis of aprostate carcinoma to the lymph-node, the DU-145 cell-line which wasderived from metastasis of prostate carcinoma to the vertebra and thePC-3 cell line which was derived from a prostate cancer metastasis tothe brain. Protein samples from the four prostate cell-lines wereresolved by 7% SDS-PAGE mid were reacted with the affinity purifiedαTMF/ARA160 antibodies, in a Western-blot analysis.

FIG. 10 illustrates the accumulation of TMF/ARA160 in human prostatecell-lines Proteins from: BPH1 (benign cell-line, lane 1); LNCAP(androgen sensitive, prostate cancer cell-line, lane 2)); DU-145(androgen insensitive, prostate cancer cell-line, lane 3) and from PC-3(androgen insensitive, prostate cancer cell-line, lane 4) cells areshown.

TMF/ARA160 was prominently detected in the benign cell line BPH1 and inthe androgen sensitive cell line LNCAP (FIG. 10 lanes 1 and 2). However,TMF/ARA160 was barely detected in the DU-145 and PC-3 cell lines (FIG.10 lanes 3 and 4) which do not respond to androgens and are thereforeconsidered as a relatively early stage of the a prostate carcinoma.

FIG. 11 shows comparative immunohistochemistry of prostate sections:normal prostate tubules (left) and malignant tubules (right). Stainingof the internal epithelial cells is clearly evident and seen in thenormal sample (left panel), and staining is absent in the malignanttissue tubules (right panel).

Example Three

Downregulation of the Cellular Level of Fer Protein, Growth Arrest andDeath of Prostate Cancer Cells in Culture by siRNA Transfection In VitrosiRNA Sequences:

The target sequences in the fer mRNA were selected and siRNA duplexesdesigned according to the guidelines described hereinabove. Two separatesiRNA duplexes were used and were obtained from commercial sources(Dharmacon, Lafayette, Colo.). The first target sequence (SEQ ID NO: 1)was (5′-) AAAGAAAUUUAUGGCCCUGAG (-3′) and begins 84 nucleotides from thestart codon. The siRNA duplex used for this sequence is referred to asthe 5′ siRNA. The sense siRNA oligonucleotide (SEQ ID NO:2) has thesequence (5′-) AGAAAUUUAUGGCCCUGAGdTdT (-3′) and the antisenseoligonucleotide (SEQ ID NO:3) has the sequence (5′-)CUCAGGGCCAUAAAUUUCUdTdT (-3′). The duplex formed is shown below:

                        5′ siRNA                          siRNA Duplex        A.G.A . A.A.U . U.U.A . U.G.G . C.C.C . U.G.A . G.dT.dT dT.dT .U.C.U . U.U.A . A.A.U . A.C.C . G.G.G . A.C.U . C

The second target sequence (SEQ ID NO:4) was (5′-) AAUCGCCCUAAGUUCAGUGAA(-3′) and begins 2407 nucleotides from the start codon. The siRNA duplexused for this sequence is referred to as the 3′ siRNA. The sense siRNAoligonucleotide (SEQ ID NO:5) has the sequence (5′-)UCGCCCUAAGUUCAGUGAAdTdT (-3′) and the antisense oligonucleotide (SEQ IDNO:6) has the sequence (5′-) UUCACUGAACUUAGGGCGAdTdT (-3′). The duplexformed is shown below:

                      3′ sIRNA                          sIRNA Duplex        U.C.G . C.C.C . U.A.A . G.U.U . C.A.G . U.G.A . A.dT.dT dT.dT .A.G.C . A.G.C . G.G.G . A.U.U . C.A.A . G.U.C . A.C.U . U

Protocol for Cell Line Transfection

PC3 cells (a cell line derived from a prostate cancer metastasis to thebrain) were counted and seeded on Petri dishes (diameter−6 cm) and weregrown in RPMI+10% FCA medium in 37° C. CO₂ 5% for 20-24 hours. 30 μlsiRNA (see above) from 20 μM stock solution was mixed with 300 μlOptiMEM (Gibco-Invitrogen) medium and incubated in room temperature for5 minutes. In a separate tube, 20 μl Metafectene (Biontex) was mixedwith 160 μl OptiMEM and also incubated in room temperature for 5 min.Then the contents of the 2 tubes were mixed (510 μl) and incubated for15-20 min in room temperature for production of lipid-siRNA complexes.The medium from the cells was removed and the cells covered with 129 μlOptiMEM, FCS 200 μl and 510 μl lipids—siRNA complexes. Finalconcentration of siRNA in the Petri dish is 300 nM 8 hours later 1800 μlOptimem and 200 μl FCS were added to the cells. After 72 hours the cellswere scraped and proteins extracted.

Results

FIG. 12 illustrates protein expression of various proteins (fer,TMF/ARA160 [labeled as co-act], CDK2, cyclin D1, actin, and cyclin B1respectively) in PC3 cells in culture after treatment with 5′ siRNA.Lane 1 (C) is the control lane—protein expression in untreated PC3cells, lane 2 (M)-cells treated with the transfection reagent alonewithout any siRNA, lane 3 (S)-cells treated with an siRNA duplex for anunrelated gene, lane 4 (T)-cells treated with siRNA directed to theTMF/ARA160 mRNA, and lane 5 (Fer)-cells treated with the 5′ siRNAduplexe against fer as above. Following treatment with the 5′ siRNAduplexes directed against fer mRNA, as can be seen in lane 5, there is amarked diminution in the protein levels of fer. Furthermore there isalso noted a decrease in levels of other growls promoting factors, cellcycle control proteins CDK2 and cyclin B1. Such decreases in growthpromoting factors are expected to result in the decrease in tumor cellproliferation.

FIG. 13 is a graph illustrating the percentage of viable PC3 cells inculture after 5′ siRNA treatment. Me bar on the left (a) illustrates thepercentage of viable cells in culture after treatment with 5′ siRNAdirected against fer mRNA compared to cells treated with thetransfection reagent alone (bar b on the right). As can be seen, aftertreatment with 5′ siRNA directed against fer mRNA, there was a markeddecrease in cell viability and only 35% of the cells in culture wereviable.

The growth arrest and cell death can also be seen directly in FIG. 14,which is a series of photomicrographs of PC3 cells in culture; Thephotomicrograph in panel a is of cells after 5′ siRNA treatmentresulting in downregulation of fer, and is compared to those of controlcultures (b and c) not treated With siRNA directed against fer mRNA.

Thus, it can be seen that short interfering RNAs (siRNA) directed towardthe Fer mRNA degraded fer mRNA and consequently downregulated thecellular level of the Fer protein. This led to the growth arrest of theprostate cancer cells and to their subsequent death.

Example Four

In Vivo Transfection of siRNA to Fer Leads to Diminished Tumor GrowthTumor Induction in Mice

Approximately 3*10⁶ PC3 cells in 100 μl Hank's Balanced Salt Solution(Sigma) are mixed with 100 μl Matrigel (BD Bioscience) just prior toinjection. The mixture is injected subcutaneously in CD1-nude mice fortumor induction. After 3-5 days, a pea size tumor has developed.

Preparation of siRNA for Injection:

Two different protocols for preparation of siRNA complexes for directinjection in vivo were employed with similar efficacy. The siRNAduplexes used were those described above in example three.

A. 0.5 μl TransIT-TKO transfection reagent (Mirus, Madison, Wis.) isadded to 13.33 μl OptiMEM and incubated in room temperature for 5 min.Then 0.5 μl siRNA is added to the solution for further incubation atroom temperature following addition of 35.5 μl OptiMEM to a final volumeof 50 μl.

B. 0.5 μl Mirus TransIT In Vivo is added to 0.5 μl siRNA and 49 μl ofWater for Embryo Transfer (Sigma) to a final volume of 50 μl.

Injection of siRNA into Mice

The tumor was then injected locally in 2-3 spots, with 50 μl 5′ siRNA ineither the siRNA carrier TransIT-TKO or Mirus TransIT In Vivo (Mirus) asabove. Injection was made on day 3 and the surface area of the tumormeasured daily.

Results

FIG. 15 is a graph illustrating tumor growth (surface area) in a mousefollowing 5′ siRNA treatment according to the present invention (b)compared to that in a control mouse not treated (a). As can be seen, thetreated tumor demonstrated diminished growth and its size was over 40%smaller than the control tumor after 3 weeks. Thus in vivoadministration of siRNA directed against fer mRNA resulted in aneffective treatment of the cancer explant with demonstrated decrease intumor size and growth.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification, or referenced in those mentioned, are herein incorporatedin their entirety by reference into the specification, to the sameextent as if each individual publication, patent or patent applicationwas specifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

1. A compound 21 nucleobases in length comprising SEQ ID NO:2.
 2. Acompound 21 nucleobases in length comprising SEQ ID NO:3.
 3. A shortinterfering ribonucleic acid molecule comprising a duplex molecule oftwo compounds, wherein the first compound is a compound 21 nucleobasesin length comprising SEQ ID NO:2, and the second compound is compound 21nucleobases in length comprising SEQ ID NO:3.
 4. A compound 21nucleobases in length comprising SEQ ID NO:5.
 5. A compound 21nucleobases in length comprising SEQ ID NO:6.
 6. A short interferingribonucleic acid molecule comprising a duplex molecule of two compounds,wherein the first compound is a compound 21 nucleobases in lengthcomprising SEQ ID NO:5, and the second compound is compound 21nucleobases in length comprising SEQ ID NO:6.